The organisms which cause the disease known as “coccidiosis” in chickens belong to the phylum Apicomplexa, class Sporozoa, subclass Coccidia, order Eucoccidia, suborder Eimeriorina, family Eimeriidae, genus Eimeria. Within the Eimerian genus there are many species, several of which are pathogenic in chickens. The species of major concern to the chicken industry are Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix and Eimeria brunetti. 
Coccidiosis has become a major economic problem in the chicken industry over the past several decades, mainly due to the overcrowding of chicken houses and the development of drug resistance by the parasite. The rearing of chickens under crowded conditions on a litter floor provides optimal conditions for the growth and spread of Eimeria parasites. Under such circumstances, sanitary control is impossible and the farmer must rely on the effectiveness of coccidiostat drugs. However, drugs must be kept in the feed at all times, shuttle programs must be used to avoid the appearance of drug resistance strains of Eimeria, and certain drugs have costly side effects. Furthermore, these coccidiostats also have antibacterial effects and therefore are considered to be infeed antibiotics. Recently the European Union has decided to ban the use of all in-feed antibiotics in the chicken industry including anticoccidial drugs. Thus, the only viable approach to the control of coccidiosis in the future is by vaccine development.
The Eimeria parasite undergoes a complex life cycle in the mucosa of the intestinal tract. This life cycle is very similar to that of the other hemosporidian parasites (i.e. plasmodium, babesia, etc.) except for the lack of an arthropod vector. Oocysts sporulate on the litter floor producing four sporocysts, each containing two sporozoites (thus belonging to the class sporozoa). The oocysts are ingested by the chicken, and the sporocysts are released by the mechanical grinding of the gizzard. The sporozoites are then released from the sporocysts due to the digestion of the sporocyst wall by proteolytic enzymes in the intestine. Mobile sporozoites then invade lymphocytes and go on to invade epithelial cells where the asexual cycle begins. The parasite goes through 2-4 cycles of replication and division (each species having a defined number of divisions) leading to the production of large numbers of daughter merozoites. After the final cycle of merozoite production the sexual cycle begins with the production of the macrogametocyte (female) and microgametocyte. The macrogametocyte is characterized by the production of wall forming bodies, while microgametocytes contain the components involved in the formation of microgametes, which bud off from the surface of the intracellular parasite.
Microgametes are flagellated and are responsible for the fertilization of the macrogamete. A zygote is formed which matures into the oocyst by fusion of the wall forming bodies and condensation of the nucleus. Oocysts are secreted in the feces, thus completing the cycle.
Over the past several years, native antigens from the sexual (gametocyte) stages of Eimeria maxima have been used to immunize laying hens. Offspring chicks were consequently vaccinated via maternal immunity (protective maternal antibody). Three major protective antigens have previously been identified in E. maxima gametocytes having molecular weights of 250, 82 and 56 kDa (EP Patent No. 0 256 536, U.S. Pat. No. 5,496,550, and U.S. Pat. No. 5,932,225). EP Patent No. 0 256 536, U.S. Pat. No. 5,496,550, and U.S. Pat. No. 5,932,225 are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. It was shown that these antigens are well conserved amongst Eimeria species (Wallach 1995) and can cross protect against the 3 major species that cause coccidiosis in broiler chickens, E. maxima, E. tenella and E. acervulina. More recently, it was shown that in floor pen trials, chicks from hens vaccinated with these native gametocyte antigens were protected against Eimeria under field conditions (Wallach 1996). This protection acts to lower the peak in oocyst shedding to a level which does not cause any damaging effect on the performance of the broiler chicken. Based on the above results it was concluded that these antigens are effective against coccidiosis in chickens and also have the potential for use against coccidiosis in other domestic animals including turkeys, geese, sheep, cattle, pigs and fish.
These three antigens were also characterized at the molecular level. Cell free translation experiments were carried out to identify the RNA molecules that encode them (Mencher er al.). cDNA molecules that encode these antigens were cloned by immunoscreening of a cDNA library made in the expression vector lambda zap (4, U.S. Pat. No. 5,932,225). By this approach, the gene encoding the 250 kDa antigen was cloned and partially sequenced. The clone pEM 250/14 was partially sequenced in U.S. Pat. Nos. 5,932,225 and 5,496,550. FIG. 8a of the subject application reproduces FIG. 11 of U.S. Pat. Nos. 5,932,225 and 5,496,550, which portrays the DNA sequence of the first 293 nucleotides of clone pEM 250/14. FIG. 8b of the subject application reproduces FIG. 12 of U.S. Pat. Nos. 5,932,225 and 5,496,550, which shows the DNA sequence of the last 196 nucleotides of clone pEM 250/14. Also, in U.S. Pat. Nos. 5,932,225 and 5,496,550, the putative genes encoding the 56 and 82 kDa antigens were cloned and sequenced.
Subsequently, Fried et al. sequenced the entire pEM 250/14 clone and found that the antigen had a molecular weight of 230 kDa rather than 250 kDa as had been previously thought. Fried et al. found that the 230 kDa gene contains highly repetitive motifs and that these repeats are contained throughout the entire gene (Fried et al.). This clone was expressed in bacteria using the pATH plasmid vector and it was shown that it is recognized by convalescent chicken sera taken 14 days post infection with E. maxima. Finally, it was shown that this gene is expressed only in the macrogametocyte stage and by immunofluorescence was found to be located in the wall forming bodies of the macrogamete (Fried et al.).
cDNA clones encoding the 56 and 82 kDa antigens were also obtained by screening the library with polyclonal antibodies as well as a monoclonal antibody against the 56 kDa antigen. This monoclonal antibody was previously shown to provide passive immunity to naive chicks (Wallach 1990). A few clones were obtained and analyzed. One of the clones was found to encode a small 10 kDa antigen and therefore was not the desired clone. Another clone was found to contain only a small part of the open reading frame (ORF) and by northern blotting was shown to hybridize with two mRNAs of about the expected size for the 56 and 82 kDa antigens. It was therefore concluded that this was the desired clone. Genomic libraries were then screened to obtain the full length clone. However, due to the highly repetitive GCA motifs in this clone, it was not possible to specifically isolate the full length clone. Attempts to clone the full length cDNA molecule were also not successful due to these repeats. Finally, attempts to express the partial cDNA clones in bacteria failed as well probably due to their unusual sequences and a reasonable level of gene expression was not obtained. It has previously been shown that the 56 and 82 kDa antigens are glycosylated (U.S. Pat. No. 5,932,225). This is based on their strong reactivity with Soybean lectin. Therefore, glycosylation may be required in order to obtain good expression of these genes and for proper conformation of the gene products.
In addition to the 56, 82 and 230 kDa antigens, a 14 kDa antigen obtained from highly purified fractions of oocyst walls has been proposed as a possible candidate for vaccines against coccidiosis (Eschenbacher et al.). However, this hypothesis has not been explored.
Several laboratories have been working on a subunit vaccine against coccidiosis. Most of these researchers have focused their efforts on the extracellular asexual stages of the life cycle, in particular the sporozoite and merozoite stages which are considered to be the most vulnerable to immune attack. In a previous study it was found that sporozoite extracts from E. tenella could induce in broilers protection against challenge infections against this parasite for up to 7 weeks of age (Karkhanis et al.). Work carried out using monoclonal antibodies against antigens from sporozoites of E. tenella led to the identification of a 25,000 molecular weight antigen which was cloned and sequenced (Eur. Patent publication No. 0 164 176, Dec. 11, 1985). Several other sporozoite genes were identified and their recombinant antigens or the transformed bacteria themselves were tested for protective immunity (Danforth et al.). The results indicated that these recombinants were only able to provide a relatively low level of protection against challenge infection with Eimeria and did not always prevent the appearance of significant lesions.
A vaccine using antigens from the merozoite stage has also been tested (European patent publication No. 0 135 073). Using these antigens to immunize young broiler chicks, it was once again found that the protection afforded was relatively low (Danforth et al.).
In 1993, it was found that there was a correlation between protective maternal immunity with the appearance of maternal antibodies against a 230 kDa merozoite (as opposed to gametocyte) antigen of Eimeria maxima (Smith et al.). This protection was often over 90% and was found to occur even when the maternal antibody level was relatively low (although reactivity with the 230 kDa protein remained strong). It was also found that a very small quantity of the native 230 kDa merozoite antigen cut out of an SDS-PAGE gel could induce a significant (60%) level of protective maternal immunity against infection with E. maxima in offspring chicks. Furthermore, Western blotting showed that this protein was expressed in both merozoites and sporozoites of E. maxima and is also well conserved between Eimeria species.
However, it is extremely difficult to isolate the E. maxima merozoite 230 kDa antigen on a large scale from the parasite itself. Therefore, there is a need to clone and express this antigen recombinantly. This will enable the production of the antigen for use in vaccination and to test its optimal concentration for inducing protective immunity.